The genus Brassica includes canola, one of the world's most important oilseed crops, and the most important oilseed crop grown in temperate geographies. Canola has been traditionally characterized as Brassica napus (a species derived as a result of inter-specific crosses of Brassica rapa and Brassica oleracea) in which erucic acid and glucosinolates have been eliminated or significantly reduced through conventional breeding. The majority of canola oil is in the form of vegetable oils produced for human consumption. There is also a growing market for the use of canola oil in industrial applications.
Canola is a polyploid species considered to have arisen from the hybridization of Brassica oleracea, having a diploid C genome, and Brassica rapa, having a diploid A genome. Cytogenetic investigation revealed the AA and CC genomes show a degree of relatedness, being partially homologous to one another and thought to have been derived from a common ancestor genome (Prakash and Hinata, 1980). Although technically classified as diploids, the genomes of both progenitor species contain a high percentage of regions duplicative of one another (Song et al., 1991). Genetic analysis revealed that the AA genome of Brassica rapa contributed ten chromosomes to Brassica napus, while Brassica oleracea contributed nine chromosomes from its CC genome as the maternal donor (Song et al., 1992).
The quality of edible and industrial oil derived from a particular variety of canola seed is determined by its constituent fatty acids, as the type and amount of fatty acid unsaturation have implications for both dietary and industrial applications. Conventional canola oil contains about 60% oleic acid (C18:1), 20% linoleic acid (C18:2) and 10% linolenic acid (18:3). The levels of polyunsaturated linolenic acid typical of conventional canola are undesirable as the oil is easily oxidized, the rate of oxidation being affected by several factors, including the presence of oxygen, exposure to light and heat, and the presence of native or added antioxidants and pro-oxidants in the oil. Oxidation causes off-flavors and rancidity as a result of repeated frying (induced oxidation) or storage for a prolonged period (auto-oxidation). Oxidation may also alter the lubricative and viscous properties of canola oil.
Oils exhibiting reduced levels of polyunsaturated fatty acids and increases in the level of monounsaturated oleic acid relative to conventional canola oil are associated with higher oxidative stability. The susceptibility of individual fatty acids to oxidation is dependent on their degree of unsaturation. Thus, the rate of oxidation of linolenic acid, which possesses three carbon-carbon double bonds, is 25 times that of oleic acid, which has only one double bond, and two times that of linoleic acid, which has two double bonds. Linoleic and linolenic acids also have the most impact on flavor and odor because they readily form hydroperoxides. High oleic oil (≧70% oleic) is less susceptible to oxidation during storage, frying and refining, and can be heated to a higher temperature without smoking, making it more suitable as cooking oil.
Two strategies are generally used to increase the oxidative stability of canola oil. In one approach, partial hydrogenation is used to lower linolenic acid content. Unfortunately, partial hydrogenation leads to the formation of trans-fatty acids, which have been linked to elevated levels of low-density lipoprotein cholesterol (LDL or “bad” cholesterol) in the blood, and consequently, to an increased risk of coronary heart disease. The second major strategy involves breeding programs to develop canola varieties with high oleic and low linolenic acid levels relative to conventional canola oil. High oleic and low linolenic mutants have been produced through mutagenesis (Rakow, 1973; Wong et al., 1991; Auld et al., 1992) and transgenic modification (Debonte and Hitz, 1996). Examples of commercially sold canola varieties having a fatty acid profile of C18:1 above 70% and C18:3 below 3.5% are the NEXERA® varieties, marketed by Dow AgroSciences LLC (Indianapolis, Ind.), which varieties produce NATREON® oil. One such line, AG019 (a NEXERA® variety) contains 71% to 78% oleic (C18:1) and <3% linolenic (C18:3) acid. AG019 was originally created by ethyl methanesulphonate (EMS) mutagenesis and is described in U.S. Pat. No. 6,169,190 B1 to Sernyk, assigned to the assignee of the present invention.
Current methods for producing F1 hybrid Brassica seeds have definite limitations in terms of cost and seed purity. Generally, these methods require stable, sib-incompatible and self-incompatible, nearly homozygous parental breeding lines, which parental breeding lines are available only after repeated selfing to generate inbred lines. Furthermore, inbreeding to develop and maintain the parental lines is accomplished by labor-intensive techniques, such as bud pollination, since Brassica hybrid seed production systems based on self-incompatible traits must utilize strongly self-incompatible plants. Environmental conditions during the breeding process, such as temperature and moisture, typically affect plant lipid metabolism, thus also affecting the content level of fatty acids (Harwood, 1999). Environmental variability, therefore, makes the phenotypic selection of plants less reliable. Deng and Scarth (1998) found that increase in post-flowering temperature significantly reduced the levels of C18:3 and increased C18:1. Similar results were reported in other studies (Yermanos and Goodin, 1965; Canvin, 1965).
Breeding for low linolenic varieties is particularly challenging since C18:3 content is a multi-gene trait and inherited in a recessive manner with a relatively low heritability. Genetic analysis of a population derived from the cross between “Stellar” (having a low C18:3 content (3%)) and “Drakkar” (having a “conventional” C18:3 level (9% to 10%)) indicated that the low C18:3 trait was controlled by two major loci with additive effects designated L1 and L2 (Jourdren et al., 1996b). These two major loci controlling C18:3 content were found to correspond to two fad3 (fatty acid desaturase 3) genes; one located on A genome (originating from Brassica rapa) and the other on the C genome (originating from Brassica olecera) (Jourdren et al., 1996; Barret et al., 1999).
Traits that are continuously varying due to genetic (additive, dominance, and epistatic) and environmental influences are commonly referred to as “quantitative traits.” Quantitative traits may be distinguished from “qualitative” or “discrete” traits on the basis of two factors: environmental influences on gene expression that produce a continuous distribution of phenotypes; and the complex segregation pattern produced by multigenic inheritance. The identification of one or more regions of the genome linked to the expression of a quantitative trait led to the discovery of Quantitative Trait Loci (“QTL”). Thormann et al., (1996) mapped two QTL that explained 60% of the variance for the linolenic content, while Somers et al., (1998) identified three QTL that collectively explained 51% of the phenotypic variation of C18:3 content. A three-locus additive model was also reported by Chen and Beversdorf (1990). Rücker and Röbelen (1996) indicated that several minor genes are most likely involved in the desaturation step.
Heritability for C18:3 content was estimated to be 26% to 59% (Kondra and Thomas, 1975) (where the variability of heritability is a function of genetics as opposed to environmental factors). Complexity of the inheritance of linolenic acid may be due to the fact that linolenic acid can be synthesized either from the desaturation of C18:2 or the elongation of C16:3 (Thompson, 1983).
In contrast to linolenic acid, inheritance of oleic acid is less complex, and the heritability of oleic acid is relatively high. It is reported that high oleic acid content is controlled by a major locus called fad2 (fatty acid desaturase 2) gene which encodes the enzyme responsible for the desaturation of oleic acid to linoleic acid (C18:2) (Tanhuanpaa et al., 1998; Schierholt et al., 2001). All of the functional gene copies of the fad2 gene that have been reported and mapped to date are located on the A-genome-originated linkage group N5 (Scheffler et al., 1997; Schierholt et al., 2000). Chen and Beversdorf (1990) reported that the accumulation of oleic acid was controlled by at least two segregation genetic systems, one acting on chain elongation and the other involving desaturation. Heritability for C18:1 content was estimated to be 53% to 78% (Kondra and Thomas 1975) and 94% (Schierholt and Becker, 1999), respectively. Due to the higher heritability, the expression of C18:1 content is environmentally less affected and relatively stable (Schierholt and Becker, 1999).
In NEXERA® canola germplasm, one to two genes are found to control C18:1 content and at least three genes are involved in C18:3 expression. In segregating progenies, the distribution of seed C18:3 content is continuous, thereby making it difficult to identify genotypic classes with desirable C18:3 levels. In addition, there is a low correlation in fatty acid content between greenhouse (GH) and field-grown plants, further making it challenging to reliably select GH plants with desirable levels of C18:3.
Molecular maker selection is based on genotypes and is, therefore, independent from environmental effects. Molecular markers would alleviate the problem of the unreliable selection of plants in the greenhouse attributable to the low correlation in fatty acid content between greenhouse-grown plants and field-grown plants. Significantly, molecular markers tightly linked to the genes controlling C18:1 and C18:3 content would allow early selection of plants carrying genes for high C18:1 and low C18:3. Marker-assisted selection at early stage will significantly save greenhouse space, therefore, improve the efficiency of greenhouse use, and reduce the breeding workload in the field.
More generally, molecular markers have advantages over morphological markers in that: molecular markers can be highly polymorphic while morphological markers are strictly phenotype dependent; morphological markers may interfere in the scoring of certain quantitative phenotypes while molecular markers exhibit a 1:1 relationship between genotype and phenotype (thus allowing the unambiguous scoring of all possible genotypes for a given locus); and epistatic interactions tend to limit the number of morphological markers useful in a population, while molecular markers do not interact epistatically.
Different types of molecular markers such as RAPD (random-amplified polymorphic DNA) markers (Tanhuanpaa et al., 1995; Hu et al., 1995; Rajcan et al., 1999; Jourdren et al., 1996), RFLP (restriction fragment length polymorphism) markers (Thormann et al., 1996) and SCAR (sequence-characterized amplified region) markers (Hu et al., 1999) have been identified to be associated with low C18:3 levels in Brassica napus. Molecular markers have also been identified for high C18:1 content. A RAPD marker was identified to be linked to the QTL affecting oleic acid concentration in spring turnip rape (B. rapa ssp. oleifera) and was later converted into a SCAR marker (Tanhuanpaa et al., 1996). Schierholt et al., (2000) identified three AFLP markers linked to a high oleic acid mutation in winter oilseed rape (B. napus L.). Tanhuanpaa et al., (1998) developed an allele-specific PCR marker for oleic acid by comparing the wild-type and high-oleic allele of the fad2 gene locus in spring turnip rape (B. rapa ssp. oleifera). However, most of these markers are low-throughput markers, such as RAPD, AFLP and RFLP, and are not suitable for large-scale screening through automation.
Therefore, what is needed in the art are molecular markers suitable for identifying canola plants producing a seed oil with desired levels of high oleic and low linolenic acid, which render the oil sufficiently stable for uses in various dietary and industrial applications. It would be further advantageous to map genes responsible for oleic and linolenic acid concentration and to develop high throughput PCR markers linked to high oleic and low linolenic acid content in order to facilitate the selection of these traits in oil seed crop trait introgression and breeding.